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Antibodies with immune effector activity and that internalize in endosialin-positive cellsRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Radionuclide Or Intended Radionuclide Containing; Adjuvant Or Carrier Compositions; Intermediate Or Preparatory Compositions, Attached To Antibody Or Antibody Fragment Or Immunoglobulin; DerivativeAntibodies with immune effector activity and that internalize in endosialin-positive cells description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060239911, Antibodies with immune effector activity and that internalize in endosialin-positive cells. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATION [0001] This claims benefit of U.S. Provisional Application 60/674,344, filed Apr. 22, 2005, which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION [0002] This invention relates to the use of monoclonal and polyclonal antibodies that specifically bind to and alternatively become internalized by cells expressing or bearing endosialin ("endosialin-positive cells") or induce an immune effector activity such as antibody-dependent cellular cytotoxicity. The antibodies are useful in specific delivery of pharmacologic agents to endosialin-positive cells as well as in eliciting an immune-effector activity particularly on endosialin-positive cells including but not limited to neovascular cells, endothelial precursors, tumor, tumor stromal and dysplastic cells. The invention is also related to cells expressing the monoclonal antibodies, polyclonal antibodies, antibody derivatives, such as chimeric and humanized monoclonal antibodies, antibody fragments, methods of detecting endosialin-positive cells, and methods of treating cancer and neovascular disease with the antibodies of the invention. BACKGROUND OF THE INVENTION [0003] Angiogenesis is a regulated process involving the formation of new blood vessels. It plays an essential role in normal growth, embryonic development, wound healing, and other physiological processes (Yancopoulos et al. (2000) Nature 407(6801):242-8). Moreover, de novo angiogenesis is involved in several disease states including cancer, where the formation of new "embryonic-like" blood vessels (referred to as neovascularization herein) appear that differ from normal vasculature with regards to structure and function (Hanahan and Weinberg (2000) Cell 100(1):57-70). A number of in vivo and in vitro studies have demonstrated biological differences between normal and disease-associated vasculature as determined using various model systems of angiogenesis offering the ability to develop novel anti-angiogenic compounds that can selectively inhibit vessel formation of the embryonic-type, tumor-associated endothelial-type for therapy of neovascular disease. In light of these opportunities for therapy, an intense search for potential targets that can specifically inhibit tumor and other neovascular disease-associated endothelial cell growth and function is ongoing. In an attempt to identify such targets, strategies have been designed to identify cell surface antigens of tumor stroma as well as isolate specific proteins or RNA that are expressed in neovascular endothelial cells (Rettig et al. (1992) Proc Natl. Acad Sci USA 89(22):10832-6; St. Croix et al. (2000) Science 289: 1197-1202). These strategies have identified a cell surface protein that appears to be specifically expressed in neovascular endothelial cells called endosialin (or tumor endothelial marker 1, TEM1). The use of antibodies in immunohistochemistry studies of malignant tissues have found good expression of endosialin in a number of neovascular endothelial cells in malignant tissues, tumor stromal cells, and putative endothelial leukocytes [(Brady et al. (2004) Human endosialin (tumor endothelial marker 1) is abundantly expressed in highly malignant and invasive brain tumors. J. Neuropathol Exp Neurol. 63:1274-83; Rettig et al. (1992); Conejo-Garcia et al. (2005) Vascular leukocytes contribute to tumor vascularization. Blood 105:679-81)] while expression in cell lines derived from embryonic-like endothelial cultures such as but not limited to HUVEC (Human Umbilical Vein Endothelial Cells) or HMVEC-(Neonatal Dermal Microvascular Endothelial Cells) is limited (available online through Cambrex Corp.). Analysis of antibodies that can bind to endosialin have identified a set of cells that express this antigen in endothelial cell cultures as well as a subset of dendritic-like cells in normal tissue of patients. Recently it has been shown that leukocytes contain cells that can form endothelial cells in vessels of tumors, supporting the use of antibodies in targeting these endosialin-positive cell types for therapy (Peters et al. (2005) Contribution of bone marrow-derived endothelial cells to human tumor vasculature. Nat. Med. 11:261-262). [0004] Neovascularization is associated with a number of disease states. In cancer it is believed that neovascularization is important to supply tumors with blood. In non-oncology diseases such as retinopathy and macular degeneration, uncontrolled neovascularization causes loss of sight (Wilkinson-Berka (2004) Curr Pharm Des. 10(27):3331-48; Das and McGuire (2003) Prog Retin Eye Res. 22(6):721-48). Moreover, several reports have identified a role of neovascularization in inflammatory disease (Paleolog and Miotla (1998) Angiogenesis 2(4):295-307). Methods to better define the embryonic-like endothelial and precursor cells associated with these disease states will lead to the development of novel drugs to treat these diseases. The development of antibodies that can specifically target endothelial cells associated with cancer or neovascular disease (age-related macular degeneration, retinopathy, inflammation, etc.) offers the ability to treat these diseases. [0005] A difficult problem in effective antiangiogenic and proangiogenic therapy is the inability to specifically target and kill or suppress the action of these cells via pharmaceutical compounds. An approach to get better specificity to treat cancer or neovascular disease is the use of antibodies that can target specific antigens expressed in cancer or neo-endothelial cells or precursors that are not expressed or are expressed at a lower level on normal cells. These targets can be exploited using antibodies to kill antigen-bearing cells by inhibiting the biological activity of the antigen, eliciting an immune effector function by complement-dependent cytotoxicity (CDC) and/or antibody-dependent cellular cytotoxicity (ADCC); or by delivering immuno- or radio-conjugates that, when delivered to the antigen-bearing cells, specifically kill the target cell. Finding antibodies that can specifically bind to and effectively kill antigen-bearing cells involved in tumorigenesis and neovascular-associated disease, such as inflammation, age-related macular degeneration, and retinopathy, has proven difficult for many biological compounds (Dhanabal (2005) Anti-angiogenic therapy as a cancer treatment paradigm Curr Med Chem Anti-Canc Agents 5:115-30). This has been due in part to the inability to get robust killing due to lack of immune-effector activity or lack of efficient internalization of antibodies carrying immunotoxins. [0006] In 1992, Rettig et al. described monoclonal antibodies that recognize antigens on vessels within various cancer types (Rettig et al. (1992) Proc Natl. Acad Sci USA 89(22):10832-6). One of these was designated FB5, which recognizes a .about.100 kDa protein on the surface of a neuroblastoma cell line, LA1-5s. FB5 is a murine IgG1 antibody that binds to endosialin and has been shown to recognize endothelial cells associated with a variety of different cancer types. Structural evaluation classified endosialin as a C-type lectin-like protein composed of five globular extracellular domains (including a C-type lectin domain, one domain with similarity to the Sushi/ccp/scr pattern, and three EGF repeats). The protein also contains a mucin-like region, a transmembrane segment, and a short cytoplasmic tail. The protein appears to be a glycoprotein. Carbohydrate analysis shows that the endosialin core protein has an abundance of sialylated, O-linked oligosaccharides with similarities to sialomucin-like molecules. Subsequent work combined the complementarity determining regions (CDR) of the mouse FB5 onto a human IgG1 backbone to create a humanized antibody that is able to also bind to vessels within malignant tissues and a subset of cells in HMVEC cultures. U.S. Pat. No. 5,342,757 describes the FB5 antibody that binds to a .about.100 kDa protein. [0007] Endosialin offers an opportunity to specifically target cancer and neovascular disease. Provided herein are in-out antibodies that can, in the alternative, internalize in endosialin-positive cells (e.g., for delivery of toxic conjugates) and elicit a cytotoxic effect via immune effector activity. The antibodies of the invention provide effective antibody therapies for cancer, neovascular-associated diseases such as but not limited to macular degeneration (e.g., age-related macular degeneration), retinopathy, and inflammation, and other conditions associated with endosialin. SUMMARY OF THE INVENTION [0008] The invention provides endosialin-specific antibodies that alternatively elicit a robust immune-effector function or internalize in endosialin-positive cells, referred to here as in-out anti-endosialin antibodies. As used herein, "in-out antibodies" ("in-out Abs") refer to antibodies that can alternatively elicit an immune effector activity and internalize within an antigen-presenting cell by binding to target antigen. Without wishing to be bound by any particular theory, it is believed that in-out Abs bind to the cell surface of an antigen-bearing cell and internalize after a period of time unless engaged by immune effector cells or biochemicals that are recruited to the antigen-antibody-bearing cell. Antibodies that are able to elicit an immune effector effect such ADCC or CDC and internalize have been previously described (Wolff et al. Monoclonal antibody homodimers: enhanced antitumor activity in nude mice. Cancer Res. 1993 Jun. 1; 53:2560-5), however, it is not obvious that in-out antibodies can be developed against any antigen or epitope (Kusano et al. Immunocytochemical study on internalization of anti-carbohydrate monoclonal antibodies. Anticancer Res. 1993 November-December; 13(6A):2207-12). In-out antibodies that can target endosialin have not been described previously. Endosialin-specific antibodies have been previously described but such antibodies are not known to internalize and to elicit an immune effector function on endosialin-bearing cells (Rettig et al. 1992; Brady et al. 2004). Antibodies that can target cell surface antigens do not always elicit an immune effector function upon binding to the cell surface antigen (Niwa et al. Defucosylated chimeric anti-CC chemokine receptor 4 IgG1 with enhanced antibody-dependent cellular cytotoxicity shows potent therapeutic activity to T-cell leukemia and lymphoma. Cancer Res. 64:2127-33, 2004; Kikuchi et al. Apoptosis inducing bivalent single-chain antibody fragments against CD47 showed antitumor potency for multiple myeloma. Leuk Res. 29:445-50, 2005; Scott et al. Immunological effects of chimeric anti-GD3 monoclonal antibody KM871 in patients with metastatic melanoma. Cancer Immun. February 22; 5:3, 2005). Provided herein are antibodies that bind to the cell surface antigen endosialin and alternatively elicit an immune effector activity (such as ADCC or CDC) and internalize within antigen-bearing cells. These antibodies and derivatives thereof are useful for therapy for cancer and neovascular disease. [0009] The invention provides in-out antibodies that specifically bind to endosialin. In some embodiments, the antibodies bind antigen with greater affinity and/or avidity than FB5. In some embodiments the in-out antibodies of the invention bind the same epitope, for example a conformational epitope, as that bound by FB5. In other embodiments, the in-out antibodies of the invention bind a different epitope as that bound by FB5. [0010] The antibodies of the invention may be chimeric, including, but not limited to human-mouse chimeric antibodies. The antibodies of the invention may also be humanized. The antibodies of the invention may also be fully human. The invention also provides: hybridoma cells that express the antibodies of the invention; polynucleotides that encode the antibodies of the invention; vectors comprising the polynucleotides that encode the antibodies of the invention; and expression cells comprising the polynucleotides of the invention, referred to as transfectomas. [0011] The invention also provides methods of producing an in-out antibody that specifically binds to endosialin. Some methods comprise the step of culturing the transfectoma or hybridoma cell that expresses an antibody of the invention. The antibody-producing cells of the invention may be bacterial, yeast, insect cells, and animal cells, preferably, mammalian cells. [0012] The invention further provides methods of inhibiting the growth of endosialin-positive cells. In some embodiments, such methods comprise administering to a patient with such cells a composition comprising an in-out antibody that specifically binds to endosialin. The methods may be used for the treatment of, for example, inflammatory disease, other neovascular disease, eye diseases including age-related macular degeneration and retinopathy, cancer and various dysplastic conditions, such as, but not limited to ovarian, breast, colorectal, renal and lung cancer. In preferred embodiments, the patients are human patients. In some embodiments, the antibodies are conjugated to one or more chemotherapeutic agents such as, but not limited to radionuclides, toxins, and cytotoxic and cytostatic agents. In other embodiments the antibodies are used in combination with one or more chemotherapeutic agents. In-out antibodies can be administered as a single agent, as a conjugated or unconjugated antibody, or in combination with the conjugated or unconjugated forms or another therapeutic agent. [0013] Previous attempts to develop in-out antibodies that can specifically target endosialin have not been reported. Here we describe the development of in-out antibodies that can internalize in endosialin-positive cells and, if not internalized, elicit a cytotoxic effect via an immune effector activity. [0014] Other features and advantages of the invention will be apparent from the detailed description and examples that follow. BRIEF DESCRIPTION OF THE DRAWINGS [0015] FIG. 1 shows an endosialin-specific binding by in-out antibody ES-1 by immunohistochemistry. Antibodies can be detected by western blot, ELISA using purified antigen, crude antigen, membrane preps or whole cells as well as immunostaining. Shown is an immunostaining identifying antibody that can specifically bind to endosialin antigen. Immunostainings can be formatted with purified, semi-purified, membrane preps or whole cells expressing endosialin. Shown here are whole cells. Upper panels are whole field analysis of cells under phase contrast. Lower panels are same field of cells using fluorescein-labeled secondary antibody of cells stained with ES-1 or isotype antibody. [0016] FIG. 2 shows the ability of ES-1 to elicit ADCC immune effector activity. ES-1 elicits a robust ADCC activity. FIG. 2A illustrates non-denaturing Western blot of HMVEC cells showing the ability of ES-1 antibody to bind to endosialin while null melanoma cells were negative. The lower panel of FIG. 2B shows target HMVEC cells (referred to as target) incubated with human peripheral blood lymphocytes (PBLs) alone (0 lane); or with increasing amount of with ES-1 (right bar) or control Ig (normal IgG-left bar). Cell cultures were assayed for killing by monitoring for lactate dehydrogenase (LDH) release that occurs upon cell lysis. As shown, ES-1 has ADCC activity on endosialin-expressing cells. The upper panel of FIG. 2B shows human melanoma cells that are null for endosialin incubated with human PBLs alone (0 lane); or with increasing amount of with ES-1 (right bar) or control Ig (normal IgG-left bar). Cell cultures were assayed for killing by monitoring for lactate dehydrogenase (LDH) release that occurs upon cell lysis. As shown, ES-1 has no ADCC activity on endosialin null cells. [0017] FIG. 3 shows the ability of ES-1 to internalize in endosialin-bearing cells. ES-1 internalizes in endosialin-expressing cells. FIG. 3 shows the ability of ES-1 linked to saporin (diamond) to kill cells in contrast to ES-1 antibody alone (not shown) while an isotype control antibody did not kill cells in conjugated toxin form (square). As control, cells not expressing endosialin were used and showed that ES-1 has no toxic effect in conjugated or unconjugated form (not shown). These data support the findings that ES-1 internalizes in endosialin-bearing cells. DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS [0018] The reference works, patents, patent applications, and scientific literature, including accession numbers to GenBank database sequences that are referred to herein establish the knowledge of those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter. Continue reading about Antibodies with immune effector activity and that internalize in endosialin-positive cells... 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